FastDigest MscI (MlsI), #FD1214 - Thermo Scientific
FastDigest MscI (MlsI), #FD1214 - Thermo Scientific
FastDigest MscI (MlsI), #FD1214 - Thermo Scientific
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PRODUCT INFORMATION<br />
<strong>Thermo</strong> <strong>Scientific</strong><br />
<strong>FastDigest</strong> <strong>MscI</strong> (<strong>MlsI</strong>)*<br />
<strong>#FD1214</strong> 50 µL (for 50 rxns)<br />
Lot: ________ Expiry Date: _______<br />
5'... T G G↓C C A...3'<br />
3'... A C C↑G G T...5'<br />
* <strong>FastDigest</strong> ®<br />
<strong>MscI</strong> (<strong>MlsI</strong>) is a proprietary formulation of <strong>MlsI</strong>, an isoschizomer<br />
of <strong>MscI</strong> having the same recognition and cleavage specificity.<br />
Supplied with: 1 mL of 10X <strong>FastDigest</strong> Buffer<br />
1 mL of 10X <strong>FastDigest</strong> Green Buffer<br />
Store at -20°C<br />
BSA included<br />
www.thermoscientific.com/onebio<br />
Description<br />
<strong>Thermo</strong> <strong>Scientific</strong> <strong>FastDigest</strong> enzymes are an advanced<br />
line of restriction enzymes for rapid DNA digestion. All<br />
<strong>FastDigest</strong> enzymes are 100% active in the universal<br />
<strong>FastDigest</strong> and <strong>FastDigest</strong> Green buffers and are able to<br />
digest DNA in 5-15 minutes. This enables any combination of<br />
restriction enzymes to work simultaneously in one reaction<br />
tube and eliminates the need for sequential digestions.<br />
<strong>FastDigest</strong> enzymes can be used to digest plasmid, genomic<br />
and viral DNA as well as PCR products and do not show star<br />
activity even in prolonged incubations.<br />
Enzymes used in common downstream applications such as<br />
ligation, blunting and dephosphorylation reactions also have<br />
100% activity in <strong>FastDigest</strong> and <strong>FastDigest</strong> Green Buffer.<br />
<strong>FastDigest</strong> Green Buffer includes a density reagent along<br />
with blue and yellow tracking dyes that allow for direct<br />
loading of the reaction mixtures on a gel.<br />
The blue dye of the <strong>FastDigest</strong> Green Buffer migrates with<br />
3-5 kb DNA fragments in a 1% agarose gel and has an<br />
excitation peak at 424 nm.<br />
The yellow dye of the <strong>FastDigest</strong> Green Buffer migrates faster<br />
than 10 bp DNA fragments in a 1% agarose gel and has an<br />
excitation peak at 615 nm.<br />
For applications that require analysis by fluorescence<br />
excitation <strong>FastDigest</strong> Buffer is recommended, as the dyes of<br />
the <strong>FastDigest</strong> Green Buffer may interfere with some<br />
fluorescence measurements.<br />
Rev.8
Recommended Reaction Conditions<br />
• 1X <strong>FastDigest</strong> Buffer or 1X <strong>FastDigest</strong> Green Buffer.<br />
• Incubation at 37°C.<br />
• 1 µL of <strong>FastDigest</strong> <strong>MscI</strong> (<strong>MlsI</strong>) is formulated to digest<br />
up to:<br />
– 1 µg of lambda DNA in 5 min.<br />
– 1 µg of plasmid DNA in 5 min.<br />
– 0.2 µg of PCR product in 30 min.<br />
– 1 µg of genomic DNA in 20 min, or 5 µg of genomic<br />
DNA in 60 min.<br />
Thermal Inactivation: Incubation at 80°C for 20 min.<br />
Methylation Effects on Digestion<br />
Dam: never overlaps – no effect.<br />
Dcm: may overlap – blocked.<br />
CpG: never overlaps – no effect.<br />
EcoKI: never overlaps – no effect.<br />
EcoBI: never overlaps – no effect.<br />
Compatible ends<br />
Check www.thermoscientific.com/research for the list of<br />
restriction enzymes producing compatible ends.<br />
Number of Recognition Sites in DNA<br />
λ ΦX174 pBR322 pUC57 pUC18/19 pTZ19R/U M13mp18/19<br />
18 0 1 0 0 0 1<br />
Note<br />
<strong>FastDigest</strong> <strong>MscI</strong> (<strong>MlsI</strong>) is blocked by overlapping dcm<br />
methylation. To avoid dcm methylation, use a dam –<br />
, dcm –<br />
strain such as GM2163 (#M0099).<br />
CERTIFICATE OF ANALYSIS<br />
Functional Activity Test<br />
1 µg of lambda DNA dcm – was completely digested with<br />
1 µL of the enzyme in 5 minutes at 37°C in 20 µL of<br />
reaction mixture.<br />
Ligation and Recleavage (L/R) Assay<br />
The ligation and recleavage assay was replaced with LO<br />
test after validating experiments showed LO test ability to<br />
trace nuclease and phosphatase activities with sensitivity<br />
that is higher than L/R by a factor of 100.<br />
Labeled Oligonucleotide (LO) Assay<br />
No detectable degradation of single-stranded or doublestranded<br />
oligonucleotides occured during incubation with<br />
1 µL of <strong>FastDigest</strong> <strong>MscI</strong> (<strong>MlsI</strong>) for 1 hour.<br />
Prolonged Incubation / Star Activity Assay<br />
No detectable degradation of 1 µg of lambda DNA dcm –<br />
due to nuclease contamination or star activity occurred<br />
during incubation with 1 µL of <strong>FastDigest</strong> <strong>MscI</strong> (<strong>MlsI</strong>)<br />
for 16 hours.<br />
Blue/White (B/W) Cloning Assay<br />
The B/W assay was replaced with LO test after validating<br />
experiments showed LO test ability to detect nuclease<br />
and phosphatase activities with sensitivity that equals to<br />
that of B/W test.<br />
Quality authorized by: Jurgita Zilinskiene<br />
(continued on back page)
Protocol for Fast Digestion of Different DNA<br />
1. Combine the following reaction components at room temperature in the order indicated:<br />
Plasmid DNA PCR product Genomic DNA<br />
Water, nuclease-free (#R0581) 15 µL 17 µL 30 µL<br />
10X <strong>FastDigest</strong> or 10X <strong>FastDigest</strong> Green Buffer 2 µL 2 µL 5 µL<br />
DNA 2 µL (up to 1 µg) 10 µL (~0.2 µg) 10 µL (5 µg)<br />
<strong>FastDigest</strong> ® enzyme 1 µL 1 µL 5 µL<br />
Total volume: 20 µL 30 µL 50 µL<br />
2. Mix gently and spin down.<br />
3. Incubate at 37°C in a heat block or water thermostat for 5 min (plasmid DNA), or for 30 min (PCR product), or for 20 min (genomic DNA).<br />
Optional: Inactivate the enzyme by heating for 20 min at 80°C.<br />
4. If the <strong>FastDigest</strong> Green Buffer was used in the reaction, load an aliquot of the reaction mixture directly on a gel.<br />
Note: The <strong>FastDigest</strong> Green Buffer can be used as an electrophoresis loading buffer for any DNA sample at a final 1X concentration. Higher<br />
concentrations of <strong>FastDigest</strong> Green Buffer in the sample supply excess salt concentration which may alter DNA mobility.<br />
Double and Multiple Digestion of DNA<br />
• The combined volume of the enzymes in the reaction mixture should not exceed 1/10 of the total reaction volume.<br />
• Use 1 µL of each enzyme and scale up the reaction conditions appropriately.<br />
• If the enzymes require different reaction temperatures, start with the enzyme that requires a lower temperature, then add the second<br />
enzyme and incubate at the higher temperature.<br />
Scaling up Plasmid DNA Digestion Reaction<br />
DNA 1 µg 2 µg 3 µg 4 µg 5 µg<br />
<strong>FastDigest</strong> enzyme 1 µL 2 µL 3 µL 4 µL 5 µL<br />
10X <strong>FastDigest</strong> or 10X <strong>FastDigest</strong> Green Buffer 2 µL 2 µL 3 µL 4 µL 5 µL<br />
Total volume: 20 µL 20 µL 30 µL 40 µL 50 µL<br />
Note: Increase the incubation time by 3-5 min if the total reaction volume exceeds 20 µL. Use water thermostat, air thermostats are not<br />
recommended due to the slow transfer of heat to the reaction mixture.
Recommendations for PCR product digestion<br />
• When introducing restriction enzyme sites into primers for subsequent digestion and cloning of a PCR product, refer to<br />
www.thermoscientific.com/fd, Reaction Conditions Guide, to define the number of extra bases required for efficient cleavage.<br />
• Use <strong>Thermo</strong> <strong>Scientific</strong> GeneJET PCR Purification Kit, #K0701 to purify PCR product prior digestion in following cases:<br />
− When PCR additives such as DMSO or glycerol where used, as they may affect the cleavage efficiency or cause star activity.<br />
− When PCR Product will be used for cloning. Active thermophilic DNA polymerase still present in PCR mixture may alter the<br />
ends of the cleaved DNA and reduce the ligation efficiency.<br />
Activity of DNA Modifying Enzymes in <strong>FastDigest</strong> and <strong>FastDigest</strong> Green Buffers, %<br />
<strong>Thermo</strong> <strong>Scientific</strong> FastAP <strong>Thermo</strong>sensitive Alkaline Phosphatase, #EF0651 100<br />
T4 DNA Ligase*, #EL0014 75-100<br />
Klenow Fragment, #EP0051 100<br />
T4 DNA Polymerase, #EP0061 100<br />
T4 Polynucleotide Kinase, #EK0031 100<br />
* 0.5 mM ATP (#R0441) is required for T4 DNA Ligase activity.<br />
PRODUCT USE LIMITATION<br />
This product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was not tested for use in diagnostics or for drug<br />
development, nor is it suitable for administration to humans or animals.<br />
Please refer to www.thermoscientific.com/onebio for Material Safety Data Sheet of the product.<br />
© 2012 <strong>Thermo</strong> Fisher <strong>Scientific</strong> Inc. All rights reserved. All trademarks are the property of <strong>Thermo</strong> Fisher <strong>Scientific</strong> Inc. and its subsidiaries.